CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibitionin vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as “melanoma initiating cells,” resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

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Quantifying ERK activity in response to inhibition of the BRAFV600E-MEK-ERK cascade using mathematical modelling

British Journal of Cancer ◽

10.1038/s41416-021-01565-w ◽

2021 ◽

Author(s):

Sara J. Hamis ◽

Yury Kapelyukh ◽

Aileen McLaren ◽

Colin J. Henderson ◽

C. Roland Wolf ◽

...

Keyword(s):

Drug Resistance ◽

Chemical Reactions ◽

Molecular Mechanisms ◽

Braf Inhibitor ◽

Standard Of Care ◽

Mek Inhibitor ◽

Mass Action ◽

Mass Action Kinetics ◽

Erk Activity

Abstract Background Simultaneous inhibition of multiple components of the BRAF-MEK-ERK cascade (vertical inhibition) has become a standard of care for treating BRAF-mutant melanoma. However, the molecular mechanism of how vertical inhibition synergistically suppresses intracellular ERK activity, and consequently cell proliferation, are yet to be fully elucidated. Methods We develop a mechanistic mathematical model that describes how the mutant BRAF inhibitor, dabrafenib, and the MEK inhibitor, trametinib, affect BRAFV600E-MEK-ERK signalling. The model is based on a system of chemical reactions that describes cascade signalling dynamics. Using mass action kinetics, the chemical reactions are re-expressed as ordinary differential equations that are parameterised by in vitro data and solved numerically to obtain the temporal evolution of cascade component concentrations. Results The model provides a quantitative method to compute how dabrafenib and trametinib can be used in combination to synergistically inhibit ERK activity in BRAFV600E-mutant melanoma cells. The model elucidates molecular mechanisms of vertical inhibition of the BRAFV600E-MEK-ERK cascade and delineates how elevated BRAF concentrations generate drug resistance to dabrafenib and trametinib. The computational simulations further suggest that elevated ATP levels could be a factor in drug resistance to dabrafenib. Conclusions The model can be used to systematically motivate which dabrafenib–trametinib dose combinations, for treating BRAFV600E-mutated melanoma, warrant experimental investigation.

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Molecular Targeting of HuR Oncoprotein Suppresses MITF and Induces Apoptosis in Melanoma Cells

Cancers ◽

10.3390/cancers13020166 ◽

2021 ◽

Vol 13 (2) ◽

pp. 166

Author(s):

Rebaz Ahmed ◽

Ranganayaki Muralidharan ◽

Akhil Srivastava ◽

Sarah E. Johnston ◽

Yan D. Zhao ◽

...

Keyword(s):

Cell Cycle ◽

Drug Resistance ◽

Targeted Therapy ◽

Antitumor Activity ◽

Mek Inhibitor ◽

Melanoma Cells ◽

Migration And Invasion ◽

Mutational Status ◽

The Impact

Background: Treatment of metastatic melanoma possesses challenges due to drug resistance and metastases. Recent advances in targeted therapy and immunotherapy have shown clinical benefits in melanoma patients with increased survival. However, a subset of patients who initially respond to targeted therapy relapse and succumb to the disease. Therefore, efforts to identify new therapeutic targets are underway. Due to its role in stabilizing several oncoproteins’ mRNA, the human antigen R (HuR) has been shown as a promising molecular target for cancer therapy. However, little is known about its potential role in melanoma treatment. Methods: In this study, we tested the impact of siRNA-mediated gene silencing of HuR in human melanoma (MeWo, A375) and normal melanocyte cells in vitro. Cells were treated with HuR siRNA encapsulated in a lipid nanoparticle (NP) either alone or in combination with MEK inhibitor (U0126) and subjected to cell viability, cell-cycle, apoptosis, Western blotting, and cell migration and invasion assays. Cells that were untreated or treated with control siRNA-NP (C-NP) were included as controls. Results: HuR-NP treatment significantly reduced the expression of HuR and HuR-regulated oncoproteins, induced G1 cell cycle arrest, activated apoptosis signaling cascade, and mitigated melanoma cells’ aggressiveness while sparing normal melanocytes. Furthermore, we demonstrated that HuR-NP treatment significantly reduced the expression of the microphthalmia-associated transcription factor (MITF) in both MeWo and MITF-overexpressing MeWo cells (p < 0.05). Finally, combining HuR-NP with U0126 resulted in synergistic antitumor activity against MeWo cells (p < 0.01). Conclusion: HuR-NP exhibited antitumor activity in melanoma cells independent of their oncogenic B-RAF mutational status. Additionally, combinatorial therapy incorporating MEK inhibitor holds promise in overriding MITF-mediated drug resistance in melanoma.

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miR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators

Cell Communication and Signaling ◽

10.1186/s12964-020-00601-1 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Elisabetta Vergani ◽

Matteo Dugo ◽

Mara Cossa ◽

Simona Frigerio ◽

Lorenza Di Guardo ◽

...

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Drug Sensitivity ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Mek Inhibitor ◽

Resistant Cell ◽

Melanoma Tumor ◽

Knock Out

Abstract Background Targeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance. Methods The miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy. Results miR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor. Conclusions Deregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment. Graphical Abstract

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Deregulated FASN Expression in BRAF Inhibitor-Resistant Melanoma Cells Unveils New Targets for Drug Combinations

Cancers ◽

10.3390/cancers13092284 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2284

Author(s):

Serena Stamatakos ◽

Giovanni Luca Beretta ◽

Elisabetta Vergani ◽

Matteo Dugo ◽

Cristina Corno ◽

...

Keyword(s):

Drug Resistance ◽

Metastatic Melanoma ◽

Mutant Cell ◽

Braf Inhibitor ◽

Cancer Cell Line ◽

Melanoma Cells ◽

Melanoma Progression ◽

Increased Sensitivity ◽

Resistant Cells

Metabolic changes promoting cell survival are involved in metastatic melanoma progression and in the development of drug resistance. In BRAF-inhibitor resistant melanoma cells, we explored the role of FASN, an enzyme involved in lipogenesis overexpressed in metastatic melanoma. Resistant melanoma cells displaying enhanced migratory and pro-invasive abilities increased sensitivity to the BRAF inhibitor PLX4032 upon the molecular targeting of FASN and upon treatment with the FASN inhibitor orlistat. This behavior was associated with a marked apoptosis and caspase 3/7 activation observed for the drug combination. The expression of FASN was found to be inversely associated with drug resistance in BRAF-mutant cell lines, both in a set of six resistant/sensitive matched lines and in the Cancer Cell Line Encyclopedia. A favorable drug interaction in resistant cells was also observed with U18666 A inhibiting DHCR24, which increased upon FASN targeting. The simultaneous combination of the two inhibitors showed a synergistic interaction with PLX4032 in resistant cells. In conclusion, FASN plays a role in BRAF-mutated melanoma progression, thereby creating novel therapeutic opportunities for the treatment of melanoma.

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Clinical efficacy with dabrafenib and trametinib in a T599_V600insT poorly differentiated metastatic thyroid carcinoma

BMJ Case Reports ◽

10.1136/bcr-2021-243264 ◽

2021 ◽

Vol 14 (8) ◽

pp. e243264

Author(s):

Chung-Shien Lee ◽

Emily Miao ◽

Kasturi Das ◽

Nagashree Seetharamu

Keyword(s):

Thyroid Carcinoma ◽

Kinase Inhibitors ◽

Mek Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Braf Mutations ◽

Effective Treatment Option ◽

Mitogen Activated Protein ◽

Treatment Data ◽

Poorly Differentiated

BRAF (v-raf murine sarcoma viral oncogene homolog B1) and MEK (mitogen-activated protein kinase kinase) inhibitors have been shown to improve clinical outcomes in tumours presenting with mutations in the BRAF gene. The most common form of BRAF mutation is V600E/K and has been shown to occur in thyroid cancers. Treatment data for patients harbouring less frequent BRAF mutations are limited. In vitro studies have shown that mutations in codons 599–601 increase kinase activity similar to that in V600E mutations, which suggests that BRAF and MEK inhibitors could be an effective treatment option. Here, we report a case of a patient with thyroid carcinoma harbouring a rare amino acid insertion in codon 599 of the BRAF gene (T599_V600insT) treated with a BRAF and MEK inhibitor.

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Anaplastic Thyroid Cancer

10.2310/cgso.16036 ◽

2019 ◽

Author(s):

Geeta Lal

Keyword(s):

Thyroid Cancer ◽

Kinase Inhibitors ◽

Braf Inhibitor ◽

Anaplastic Thyroid Cancer ◽

Mek Inhibitor ◽

Braf V600e ◽

Multidisciplinary Teams ◽

Neoadjuvant Radiotherapy ◽

Current State ◽

Targeted Treatments

Anaplastic thyroid cancer (ATC) is a rare thyroid malignancy with a nearly uniform poor prognosis. Most patients present with advanced disease, and optimal management requires rapid diagnosis, staging, and involvement of multidisciplinary teams. Treatment may include surgery in patients with resectable disease and adjuvant or neoadjuvant radiotherapy and chemotherapy. Improved understanding of molecular pathogenesis has allowed the assessment of tyrosine kinase inhibitors and other targeted treatments in these patients. The FDA recently approved the combination of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for the treatment of BRAF V600E mutation positive, unresectable or metastatic ATC. This review summarizes the current state-of-the-art concepts in the management of patients with ATC. This review contains 3 figures, 2 tables, and 25 references. Key words: anaplastic thyroid cancer, goals of care discussion, management, surgery, radiotherapy, chemotherapy novel therapies, NCCN and ATA guidelines

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Use of kinase inhibitors against schistosomes to improve and broaden praziquantel efficacy

Parasitology ◽

10.1017/s0031182020001250 ◽

2020 ◽

Vol 147 (13) ◽

pp. 1488-1498

Author(s):

Sujeevi S. K. Nawaratna ◽

Donald P. McManus ◽

Robin B. Gasser ◽

Paul J. Brindley ◽

Glen M. Boyle ◽

...

Keyword(s):

Drug Resistance ◽

Kinase Inhibitors ◽

Lethal Dose ◽

Alternative Therapies ◽

Potential Drug ◽

Dependent Protein Kinase ◽

Drug Of Choice ◽

Dependent Protein ◽

The Impact

AbstractPraziquantel (PZQ) is the drug of choice for schistosomiasis. The potential drug resistance necessitates the search for adjunct or alternative therapies to PZQ. Previous functional genomics has shown that RNAi inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) gene in Schistosoma adult worms significantly improved the effectiveness of PZQ. Here we tested the in vitro efficacy of 15 selective and non-selective CaMK inhibitors against Schistosoma mansoni and showed that PZQ efficacy was improved against refractory juvenile parasites when combined with these CaMK inhibitors. By measuring CaMK activity and the mobility of adult S. mansoni, we identified two non-selective CaMK inhibitors, Staurosporine (STSP) and 1Naphthyl PP1 (1NAPP1), as promising candidates for further study. The impact of STSP and 1NAPP1 was investigated in mice infected with S. mansoni in the presence or absence of a sub-lethal dose of PZQ against 2- and 7-day-old schistosomula and adults. Treatment with STSP/PZQ induced a significant (47–68%) liver egg burden reduction compared with mice treated with PZQ alone. The findings indicate that the combination of STSP and PZQ dosages significantly improved anti-schistosomal activity compared to PZQ alone, demonstrating the potential of selective and non-selective CaMK/kinase inhibitors as a combination therapy with PZQ in treating schistosomiasis.

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MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia

Scientific Reports ◽

10.1038/s41598-019-54901-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

María Luz Morales ◽

Alicia Arenas ◽

Alejandra Ortiz-Ruiz ◽

Alejandra Leivas ◽

Inmaculada Rapado ◽

...

Keyword(s):

Drug Resistance ◽

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Resistance Pathway ◽

Acute Myeloid

AbstractFMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

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Bioactive Compounds from Seaweed with Anti-Leukemic Activity: A Mini-Review on Carotenoids and Phlorotannins

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557519666190311095655 ◽

2020 ◽

Vol 20 (1) ◽

pp. 39-53 ◽

Cited By ~ 1

Author(s):

Tânia P. Almeida ◽

Alice A. Ramos ◽

Joana Ferreira ◽

Amaya Azqueta ◽

Eduardo Rocha

Keyword(s):

Drug Resistance ◽

Bioactive Compounds ◽

Myeloid Leukemia ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

First Line ◽

In Vivo Models ◽

Few Data

: Chronic Myeloid Leukemia (CML) represents 15-20% of all new cases of leukemia and is characterized by an uncontrolled proliferation of abnormal myeloid cells. Currently, the first-line of treatment involves Tyrosine Kinase Inhibitors (TKIs), which specifically inhibits the activity of the fusion protein BCR-ABL. However, resistance, mainly due to mutations, can occur. In the attempt to find more effective and less toxic therapies, several approaches are taken into consideration such as research of new anti-leukemic drugs and “combination chemotherapy” where different drugs that act by different mechanisms are used. Here, we reviewed the molecular mechanisms of CML, the main mechanisms of drug resistance and current strategies to enhance the therapeutic effect of TKIs in CML. Despite major advances in CML treatment, new, more potent anticancer drugs and with fewer side effects are needed. Marine organisms, and particularly seaweed, have a high diversity of bioactive compounds with some of them having anticancer activity in several in vitro and in vivo models. The state-of-art suggests that their use during cancer treatment may improve the outcome. We reviewed here the yet few data supporting anti-leukemic activity of some carotenoids and phlorotannins in some leukemia models. Also, strategies to overcome drug resistance are discussed, particularly the combination of conventional drugs with natural compounds.

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c-Abl Kinase Inhibitors Overcome CD40-Mediated Drug Resistance in CLL.

Blood ◽

10.1182/blood.v110.11.3078.3078 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3078-3078 ◽

Cited By ~ 1

Author(s):

Delfine Y. Hallaert ◽

Annelieke Jaspers ◽

Carel J. van Noesel ◽

Marinus H.J. Van Oers ◽

Arnon P. Kater ◽

...

Keyword(s):

Drug Resistance ◽

Molecular Basis ◽

Kinase Inhibitors ◽

Lymphocytic Leukemia ◽

Recent Analysis ◽

Combination Strategies ◽

Cd40 Stimulation ◽

Erk Inhibition ◽

Erk Activity

Abstract In chronic lymphocytic leukemia (CLL), proliferation centers reside in lymph node (LN) and possibly also bone marrow, where the environment protects CLL cells from apoptotic and cytotoxic triggers. This protective milieu may well contribute to the lack of curative chemotherapy in CLL, and our recent analysis of the distinct profiles of apoptosis regulators in CLL LN versus peripheral blood supports this notion (Smit et al., Blood 2007, 109: 1660). The aim of the present study was to define the molecular basis for the increased drug resistance and to search for novel strategies to circumvent it. To mimic the situation in CLL LN, we applied prolonged in vitro CD40 stimulation of CLL cells, which results in strong upregulation of anti-apoptotic Bcl-xL and Mcl-1. Moreover, we now also report a gradual reduction of Bim at the protein level, further contributing to the anti-apoptotic profile. Using specific inhibitors (PD98059 and MG132), we found that the decrease in Bim is due to ERK-mediated phosporylation and subsequent proteasomal degradation. ERK inhibition during CD40 triggering abrogated the decrease in Bim levels, but did however not re-establish sensitivity to various drugs (fludarabine, Velcade, Roscovitine). In chronic myeloid leukemia (CML), changes in Bcl-xL, Mcl-1 and Bim levels similar to those observed in our CLL/CD40 system are known to depend on BCR-Abl signaling. Therefore, we next applied c-Abl inhibitors Gleevec or Dasatinib in conjunction with CD40. Both drugs caused a profound reversal of most protective CD40 effects; ERK activity, Bim, Bcl-xL and Mcl-1 levels as well as sensitivity to subsequent drug treatment were restored to pre-CD40 values. These effects also occurred in CLL samples with dysfunctional p53 (n=3). Importantly, in CLL LN samples we also found strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels, suggesting that there might be a c-Abl dependent survival pathway in proliferation centers. These data provide a molecular basis for combination strategies that could target refractory niches in CLL, using therapeutics that function independently of p53.

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